Summary The mammalian central nervous system supports a multitude of cognitive and behavioral functions through coordinated action of different neural circuits that are composed of diverse sets of differentiated cell types, including both neurons and non-neuronal cells. Glial cells are essential constituents of the brain and play vital roles in the development and function of the neural circuits. Compared to neurons, however, glial cells have been understudied in the past. One significant hurdle is the limited tools and technologies to precisely target and manipulate these cells in vivo. Novel AAV-based CRISPR technologies have recently been developed in the Chen lab that enables high-throughput, direct in vivo gene editing in the mouse brain. Furthermore, a new single-cell RNA sequencing (scRNA-seq) method, Act-seq, has recently been developed in the Hong lab that enables faithful characterization of cell types and detection of rapid transcriptional changes at the level of single cells. The proposed project will build upon the strong expertise of the two labs to further develop innovative technologies to provide a powerful toolbox for editing, labeling, manipulating, and profiling of specific types of glial cells in vivo. Aim 1 will develop, optimize and validate AAV-based CRISPR technology for direct in vivo tagging, labeling, and functional manipulation of glial cells in the mammalian brain. Aim 2 will develop, optimize, and validate single-cell RNA-seq technology for faithful transcriptional profiling of glial cell types and accurate detection of their transcriptional changes in response to physiological and genetic perturbations. Finally, Aim 3 will combine CRISPR technology with scRNA-seq for multiplexed gene editing and transcriptome profiling of glial cells in vivo at single-cell level. The development and combination of the above two new technologies will empower the versatile targeting, identification, and manipulation of glial cells and open multiple new directions. Establishment of these tools will offer new capabilities to rapidly gain insights into glial cells' compositions, functions, and interplay with neurons for better dissection of neural circuits and understanding of complex behaviors.